TECHNICAL FIELD
[0001] The present invention relates to a purification method for oxidatively damaged guanine
nucleosides, particularly a purification method for 8-hydroxydeoxyguanosines (hereunder,
abbreviated 8-OH-dG), a measuring method therefor, and an analyzer for performing
such.
BACKGROUND ART
[0002] Recently, the effect of active oxygen in vivo has been well researched. Normally,
active oxygen acts as a defense system when a foreign body is invading into a living
organism. However, if excessive active oxygen is generated due to food additives (carcinogen),
air pollution, smoking, stress, and the like, it causes DNA damage and produces 8-OH-dG
which is a kind of oxidative DNA damaged product. This 8-OH-dG is considered to induce
mutation and play an important role in the carcinogenesis process. Moreover, attention
has been paid to the active oxygen as a causative factor not only of carcinogenesis
but also of various disorders or aging.
[0003] Therefore, by knowing the amount of active oxygen in vivo, individual carcinogenesis
risk evaluation, prediction and diagnosis of various disorders, evaluation of the
degree of aging or general health can be performed.
[0004] However, since the active oxygen in vivo is unstable, it is difficult to directly
detect this. Therefore, as an index of active oxygen, it has been proposed to measure
the 8-OH-dG produced by the active oxygen.
[0005] Analysis methods of 8-OH-dG reported so far can be largely classified into six types,
including; (1) a method of analyzing by HPLC-ECD, a fraction which was purified by
an affinity column having antibodies against 8-OH-dG, (2) a method of connecting three
or four columns and using a column switching method to finally detect 8-OH-dG by HPLC-ECD,
(3) a method of directly analyzing urine by the ELISA method, (4) a method by a GC-MS
(requiring an internal standard substance), (5) a method by an LC-MS-MS (requiring
an internal standard substance), and (6) a method of connecting a multifunction column
(a gel filter column having both functions of reverse phase column and cation-exchange
column) and a reverse phase column through a sampling injector (an apparatus which
collects a specific fraction and injects into columns after mixing), so as to detect
by ECD.
[0006] However, in method (1), due to the low recovery rate, it is required to use a radioactive
internal standard substance.
[0007] In method (2), the pretreatment is complicated. Moreover, it is difficult to determine
the timing for switching valves and impurities are often found in the vicinity of
the peak of 8-OH-dG in many cases. Furthermore, since not only a large amount of eluent
and washing solution is required but also a large amount of poisonous effluent is
generated, it is not preferable from an environmental aspect.
[0008] In method (3), since there is a problem in the specificity, the reproducibility is
inferior.
[0009] In methods (4) and (5), since the measuring equipment is expensive, it is not preferable
in terms of economic efficiency. Furthermore, since the recovery rate is unstable,
internal standard substances are required, however they are difficult to obtain.
[0010] In method (6), since the analyzing time per one specimen is as long as 165min, the
mass treatment by continuous operation is difficult. Furthermore, when analyzing by
chromatography, impurities overlapping the peak of 8-OH-dG are very likely to be detected.
Therefore, the measurement accuracy is insufficient in any of the methods. An object
of the present invention is to provide a purification method for oxidatively damaged
guanine nucleosides, particularly 8-OH-dG, having high accuracy and reproducibility,
and also for which consideration is given to economic efficiency and environmental
aspects, a measuring method therefor, and an analyzer for performing such.
DISCLOSURE OF INVENTION
[0011] The present inventor found that oxidatively damaged guanine nucleosides such as 8-OH-dG,
8-hydroxyguanosine (ribonucleoside) (hereunder, abbreviated 8-OH-rGuo), or the like
can be specifically absorbed and recovered by using an anion-exchange column. Furthermore,
regarding 8-OH-dG, he found that 8-OH-dG can be accurately fractionated by using 8-OH-rGuo
as an internal standard marker, consequently 8-OH-dG can be measured with high accuracy
and reproducibility, and in addition, it is superior in terms of economic efficiency
and environmental aspects. From these findings he has completed the present invention.
[0012] That is, the purification method for oxidatively damaged guanine nucleosides of the
present invention is a purification method for oxidatively damaged guanine nucleosides
generated as a result of guanine damage in DNA or RNA, comprising a first purification
step for purifying oxidatively damaged guanine nucleosides contained in a sample by
anion-exchange chromatography. Moreover, the oxidatively damaged guanine nucleoside
is preferably 8-OH-dG. The purification method for 8-OH-dG of the present invention
is a purification method for 8-hydroxydeoxyguanosines (8-OH-dG) contained in a sample,
wherein 8-hydroxyguanosines (ribonucleosides) (8-OH-rGuo) are previously added to
the sample as an internal standard marker for 8-OH-dG so as to purify it. The purification
method for 8-OH-dG of the present invention is a purification method for 8-hydroxydeoxyguanosines
(8-OH-dG) contained in a sample, wherein 8-hydroxyguanosine (ribonucleosides) (8-OH-rGuo)
is previously added to the sample, comprising a first purification step for purifying
the sample by anion-exchange chromatography, and a second purification step for further
purifying the fraction containing 8-OH-dG obtained in the first purification step
by reverse phase chromatography. In the purification method for oxidatively damaged
guanine nucleosides, the sample is preferably urine. Moreover, in the purification
method for 8-OH-dG, the sample is preferably urine. The measuring method for oxidatively
damaged guanine nucleosides of the present invention comprises a measuring step for
measuring purified oxidatively damaged guanine nucleosides obtained by the purification
method. The measuring method for 8-OH-dG of the present invention comprises a measuring
step for measuring purified 8-OH-dG obtained by the purification method. In the measuring
method for 8-OH-dG of the present invention, the purified 8-hydroxydeoxyguanosines
(8-OH-dG) are measured in anion-exchange chromatography in the order of; (1) peak
recognition of ribonucleosides 8-OH-rGuo, (2) starting of 8-OH-dG fractionation after
a fixed time, (3) finishing of 8-OH-dG fractionation after a fixed time, and (4) optionally
mixing 8-OH-dG fraction, and then injected into a reverse phase column.
[0013] The analyzer of the present invention is an apparatus for purifying and measuring
8-hydroxydeoxyguanosines (8-OH-dG), comprising; an anion-exchange column (HPLC-1)
which specifically absorbs 8-OH-dG contained in a sample, a UV detector which detects
an elution position of 8-hydroxyguanosine (ribonucleoside) (8-OH-rGuo), a reverse
phase column (HPLC-2) which further purifies the fraction containing 8-OH-dG obtained
from the anion-exchange column (HPLC-1), and a detector which measures the purified
8-OH-dG obtained from the reverse phase column (HPLC-2).
[0014] The control program of the present invention is a program for controlling a process
for recovering 8-hydroxydeoxyguanosines (8-OH-dG) contained in a sample by column
chromatography, which executes on a computer processes for: receiving a peak signal
of a marker (8-OH-rGuo) previously added to the sample from a UV detector; outputting
a signal to open a valve connected to a sampler, during 8-OH-dG elution after a fixed
time; starting fractionation; and outputting a fractionation termination signal after
another fixed time; and then outputting a signal to inject the obtained 8-OH-dG fraction
into a second purifying column; thereby purifying and recovering a detected substance
(8-OH-dG) eluted from the column.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015]
FIG. 1 is a schematic diagram showing an embodiment of an apparatus for purifying
and measuring 8-OH-dG.
FIG. 2 is a schematic diagram showing an embodiment of an apparatus for purifying
and measuring 8-OH-dG.
FIG. 3 shows an example of a separation pattern of a mixture of urine, 8-OH-dG and
8-OH-rGuo, using an anion-exchange column (HPLC-1), showing a positional validation
of the markers.
FIG. 4 shows an example of a separation pattern of human urine using an anion-exchange
column (HPLC-1).
FIG. 5 shows an example of a separation pattern of human urine using a reverse phase
column (HPLC-2).
FIG. 6 shows an example of a separation pattern of human urine using an anion-exchange
column (HPLC-1).
FIG. 7 shows an example of a separation pattern of human urine using a reverse phase
column (HPLC-2).
FIG. 8 shows an example of a separation pattern of rat urine using a reverse phase
column (HPLC-2).
FIG. 9 shows an example of a separation pattern of rat urine using a reverse phase
column (HPLC-2).
BRIEF DESCRIPTION OF THE REFERENCE SYMBOLS
[0016]
- 11.
- Anion-exchange column (HPLC-1)
- 12.
- Reverse phase column (HPLC-2)
- 13.
- Detector
- 14.
- UV detector
- 15.
- Switching valve
- 16.
- Switching valve
- 17.
- Automatic sampler
- 27.
- Sampling injector
BEST MODE FOR CARRYING OUT THE INVENTION
[0017] The present invention is a purification method for accurately measuring oxidatively
damaged guanine nucleosides being the index of active oxygen, particularly 8-OH-dG,
a measuring method therefor, and an analyzer for performing such, in order to evaluate
the amount of active oxygen in vivo.
[0018] The oxidatively damaged nucleoside including 8-OH-dG is generated as a result of
DNA or RNA damage by active oxygen (oxygen radical) and the like in vivo, and is used
as the index of active oxygen. The oxidatively damaged nucleosides besides 8-OH-dG
include 2-hydroxydeoxyadenosine (2-OH-dA), 5-hydroxydeoxycytidine (5-OH-dC), 5-formyldeoxyuridine
(5-CHO-dU), 8-OH-rGuo, and the like. These oxidatively damaged nucleosides are excreted
out of the organism as undesired substance in urine. Moreover, among them, oxidatively
damaged guanine nucleosides such as 8-OH-dG, 8-OH-rGuo or the like are negatively
charged so that they are easily purified and recovered by an anion-exchange column
described in the following paragraph. Among them, it is preferable to use specifically
8-OH-dG for the index of active oxygen. The oxidatively damaged guanine nucleoside
in the present application is generated as a result of guanine damage in DNA or RNA
by active oxygen (oxygen radical), and oxidatively damaged means hydroxylation.
[0019] Samples used for the purification method and the measuring method for oxidatively
damaged guanine nucleosides (including 8-OH-dG) of the present invention includes
all biological samples such as urine, serum, cerebrospinal fluid, saliva, the medium
after culturing cells, and the like. Among them, urine is particularly preferable
since it is easy to collect and the oxidatively damaged guanine nucleosides are stable
therein.
[0020] Hereunder is a description of a purification method, a measuring method, and an analyzer
for performing such, according to the present invention, in relation to 8-OH-dG.
[0021] An apparatus for purifying and measuring 8-OH-dG according to an embodiment of the
present invention comprises; an anion-exchange column (HPLC-1) which specifically
absorbs 8-OH-dG, a UV detector which detects 8-OH-rGuo being an index of the elution
position of 8-OH-dG, a reverse phase column (HPLC-2) which further purifies the fraction
containing 8-OH-dG obtained by the anion-exchange column (HPLC-1), and a detector
which measures the purified 8-OH-dG obtained by the reverse phase column (HPLC-2).
FIG. 1 is a schematic diagram showing an example of an analyzer. In the diagram, reference
symbol 11 is an anion-exchange column (HPLC-1). This is connected to a reverse phase
column (HPLC-2) 12 via a UV detector 14 and a column switching valve 16. Moreover,
upstream of the anion-exchange column (HPLC-1) 11 is connected a column switching
valve 15 to which is connected an automatic sampler 17 for injecting samples.
[0022] Furthermore, pumps 21, 22 and 23 are provided for sending solution A, B and C to
the respective columns. The solution A and B are eluents for eluting molecules absorbed
in the columns (an eluent used for the anion-exchange column (HPLC-1) 11 is solution
A and an eluent used for the reverse phase column (HPLC-2) 12 is solution B). The
solution C is a washing solution for washing a guard column (filled with an anion-exchange
resin which is the same as used in the anion-exchange column (HPLC-1) 11) connected
to the column switching valve 15. The pump 21 is connected to the automatic sampler
17. The pump 22 is connected to the column switching valve 16. The pump 23 is connected
to the column switching valve 15.
[0023] In FIG. 1, instead of the automatic sampler 17, a sampling injector ("231XL" manufactured
by Gilson) having a function to automatically operate the column switching valve 16
by peak detection of 8-OH-rGuo, may be used.
[0024] In order to perform this method, a new program was loaded into the 231XL and the
measurement performed. This program performs (1) peak recognition of ribonucleosides
8-OH-rGuo, (2) starting of 8-OH-dG fractionation at a fixed time, (3) finishing of
8-OH-dG fractionation at a fixed time, and (4) injection into the HPLC-2 (refer to
FIG. 6). In more detail, these functions are realized by the following flows.
(1) A sample is injected into the HPLC-1 by the 231XL.
(2) The system is kept standing-by for a preset time (T1).
(3) The 231XL starts monitoring the signal from the UV detector.
(4) The system is kept standing-by until exceeding the preset UV level (peak detection).
(5) After the peak detection, the system is kept standing-by for a preset time (T2).
Then a contact signal is output to a valve so as to start fractionation inside the
loop.
(6) After a preset time (T3), the contact signal is output to the valve. Then, fractionation
is finished and at the same time the fraction inside the loop is injected into HPLC-2.
[0025] As described later, according to the sampling injector ("231XL" manufactured by Gilson),
the fractionation range (time) of 8-OH-dG is automatically determined based on the
relative position with respect to 8-OH-rGuo. Therefore it is not necessary to preset
the fractionation range (time) of 8-OH-dG.
[0026] Since the abovementioned anion-exchange column (HPLC-1) 11 specifically absorbs 8-OH-dG
contained in the sample, the recovery rate is very high and almost all impurities
can be removed, so that fractions with less impurities can be obtained. Moreover,
as described above, according to the anion-exchange column (HPLC-1) 11, negatively
charged oxidatively damaged guanine nucleosides such as 8-OH-rGuo or the like can
be easily purified and recovered. The anion-exchange column (HPLC-1) 11 is not specifically
limited provided an anion-exchange resin is used for the filler. Examples of specific
filler include styrenedivinylbenzene polymer with quaternary ammonium group, polyhydroxymethacrylate
polymer with quaternary ammonium group, and the like. Moreover, examples of commercial
filler include Aminex HPX-72S (manufactured by Bio-Rad), Shodex column filler (manufactured
by Showa Denko K.K.), MCI GEL CA08F (manufactured by Mitsubishi Chemical Industries
Ltd., Hamilton RCX-10), and the like.
[0027] Moreover, the particle diameter of the anion-exchange resin is preferably from 7
to 12µm.
[0028] The internal diameter of the column which is filled with the anion-exchange resin
is not specifically limited, however preferably this is from about 1mm to 1.5mm. In
the case where the internal diameter of the column is from 2.0 to 4.6mm as shown in
FIG. 2, it is preferable to use a sampling injector 27 ("233XL" manufactured by Gilson,
or the like) connected to the column switching valve 16 so that the fraction containing
8-OH-dG is automatically injected into the reverse phase column (HPLC-2) 12. In order
to perform this method, a new program was loaded into the 233XL and the measurement
performed. This program performs (1) peak recognition of ribonucleosides 8-OH-rGuo,
(2) starting of 8-OH-dG fractionation at a fixed time, (3) finishing of 8-OH-dG fractionation
at a fixed time, (4) mixing of 8-OH-dG fraction, and (5) injection into the HPLC-2.
In more detail, these functions are realized by the following flows.
(1) A sample is injected into the HPLC-1 by the 233XL.
(2) The system is kept standing-by for a preset time (T1).
(3) The 233XL starts monitoring the signal from the UV detector.
(4) The system is kept standing-by until exceeding the preset UV level (peak detection).
(5) After the peak detection, the system is kept standing-by for a preset time (T2).
Then fractionation inside the 233XL vial tubes is started.
(6) After a preset time (T3), the fractionation is finished.
(7) The obtained fraction is stirred by drawing and discharging.
(8) The fraction is partly injected into the HPLC-2.
[0029] The length of the column which is filled with the anion-exchange resin is not specifically
limited. However the column may be shortened according to the particle diameter of
the anion-exchange resin, the exchange capacity, or the like, so as to shorten the
analysis time.
[0030] The abovementioned UV detector 14 monitors the fraction eluted out from the anion-exchange
column (HPLC-1) 11, and detects the elution position of 8-OH-dG contained in the sample.
In this manner, by monitoring the elution position of 8-OH-rGuo by the UV detector
14, the elution time of 8-OH-dG can be obtained. Together with the above, by operating
the column switching valve 16, the fraction containing 8-OH-dG can be reliably collected.
[0031] The abovementioned reverse phase column (HPLC-2) 12 further purifies the fraction
containing 8-OH-dG obtained from the anion-exchange column (HPLC-1). This column is
not specifically limited as long as it has the property of a reverse phase column.
Examples of commercial products include YMC-Pack ODS-AM (S-5µm) (manufactured by YMC
Co., Ltd.), Shiseido Capcell Pac C18 MG (S-5µm) (manufactured by Shiseido Co. Ltd.),
and the like.
[0032] The abovementioned detector 13 measures the purified 8-OH-dG obtained by the reverse
phase column (HPLC-2), and is provided downstream of the reverse phase column (HPLC-2)
12. For the detector 13, an Electrochemical detector (ECD), a liquid chromatography
mass spectrometry (LCMS), and the like may be used. Moreover, concerning the Electrochemical
detector (ECD), the peak of 8-OH-dG appears in a characteristic ratio by selecting
two kinds of preset voltages, so that the peak can be identified to be 8-OH-dG.
[0033] Moreover, the analyzer for purifying and measuring 8-OH-dG according to the embodiment
of the present invention can treat a large amount of samples by continuous operation.
In this case, the washing solution (solution C) of the guard column 35 is preferably
a composition of 0.5M ammonium sulfate : acetonitrile = about 7:3.
[0034] As described above, according to the 8-OH-dG analyzer according to the embodiment
of the present invention, the anion-exchange column (HPLC-1) 11 specifically absorbs
8-OH-dG contained in the sample, and removes almost all impurities contained in the
sample at once. Moreover, based on the elution position of 8-OH-rGuo detected by the
UV detector 14, the purified 8-OH-dG can be reliably fractionated so that it can be
superior in the recovery rate and the reproducibility. Furthermore, by continuous
operation a large amount of samples can be treated. Since the analyzer is relatively
low in price, it is also superior in terms of economic efficiency.
[0035] Hereunder is a description of the purification method and the measuring method for
8-OH-dG using the analyzer shown in FIG. 1.
(Determination of fractionation range (time))
[0036] A mixture of 8-OH-dG, 8-OH-rGuo, and urine was injected into the analyzer shown in
FIG. 1 so as to previously determine the fractionation range of 8-OH-dG. FIG. 3 shows
an example of a separation pattern of the mixture of 8-OH-dG, 8-OH-rGuo, and urine,
showing a positional validation of 8-OH-rGuo being an internal standard marker of
8-OH-dG. In this manner, by previously determining the fractionation range, an accurate
elution time for 8-OH-dG can be obtained. Together with the above, by setting so as
to operate the column switching valve 16, the fraction containing 8-OH-dG can be reliably
collected. As described above, in FIG. 1, if the sampling injector (231XL) is used
instead of the automatic sampler 17, since the fractionation range (time) of 8-OH-dG
is automatically determined by the peak detection based on the relative position with
respect to 8-OH-rGuo, it is not necessary to preset the fractionation range (time)
of 8-OH-dG.
(Purification Method)
[0037] The purification method for 8-OH-dG of the present invention comprises a first purification
step for purifying the sample by anion-exchange chromatography. Moreover, as described
above, negatively charged oxidatively damaged guanine nucleosides such as 8-OH-rGuo
or the like as well as 8-OH-dG can be easily purified and recovered by the anion-exchange
chromatograph.
[0038] The elution conditions in the first purification step are preferably such that, when
the column temperature is from 60 to 65°C and the internal diameter of the column
is 1mm, the flow rate is from 17 to 20µl/min.
[0039] In the purification method for 8-OH-dG of the present invention, it is preferable
to previously add 8-OH-rGuo to the sample as the internal standard marker for 8-OH-dG,
so as to purify it. If the 8-OH-rGuo is previously added to the sample, the 8-OH-dG
is eluted at a fixed time after the elution of the 8-OH-rGuo. Accordingly, by monitoring
the elution position of 8-OH-rGuo by the UV detector 14, the accurate elution position
(time) of 8-OH-dG can be obtained, so that the fraction containing 8-OH-dG can be
reliably collected.
[0040] Moreover, in the purification method for 8-OH-dG of the present invention, it is
preferable to previously add 8-OH-rGuo to the sample as the internal standard marker
for 8-OH-dG so as to perform the first purification step by anion-exchange chromatography,
and to further purify the fraction containing 8-OH-dG obtained in the first purification
step (second purification step).
[0041] For the second purification step, it is preferable to purify by reverse phase chromatography.
Since the eluent (solution B) used for the reverse phase chromatography, the temperature
condition, and the like vary depending on the reverse phase column (HPLC-2) 12 to
be used, these are appropriately determined. For example, in the case where human
urine is analyzed using the YMC-Pack ODS-AM (S-5µm) (manufactured by YMC Co., Ltd.)
as the reverse phase column, preferably, the column temperature is about 40°C, and
the flow rate is about 0.9ml/min.
(Measuring Method)
[0042] The measuring method of the present invention comprises a measuring step for measuring
the amount of the purified 8-OH-dG obtained by the purification method described above,
wherein the abovementioned Electrochemical detector (ECD), a liquid chromatography
mass spectrometry (LCMS), and the like may be used for measuring the amount of the
purified 8-OH-dG. The measuring method is applicable for measuring the purified oxidatively
damaged guanine nucleosides such as 8-OH-rGuo or the like as well as 8-OH-dG.
[0043] Moreover, in the case of continuous operation, both in the apparatus comprising the
automatic sampler 17 as shown in FIG. 1 and in the apparatus comprising the sampling
injector 27 as shown in FIG. 2, the elution position of 8-OH-dG is preferably checked
regularly.
[0044] As described above, according to the purification method for the oxidatively damaged
guanine nucleosides of the present invention, the purified oxidatively damaged guanine
nucleosides can be obtained with high recovery rate. Furthermore, since the flow rate
of the anion-exchange column (HPLC-1) in the first purification step is very low,
the consumption of the eluent (solution A and solution B) and the washing solution
(solution C) is extremely small, and the amount of the effluent after purification
is small, so that the method is also preferable from the aspect of environmental protection.
According to the purification method for 8-OH-dG of the present invention, the purified
8-OH-dG can be reliably fractionated and a fraction with less impurities near the
peak of 8-OH-dG can be obtained. Moreover, even in the case of continuous operation,
by the peak detection of 8-OH-rGuo, it becomes possible to correspond to the displacement
of the fraction range of each sample, so that the fraction containing 8-OH-dG can
be reliably collected. Moreover, since the measuring method of the present invention
measures the purified oxidatively damaged guanine nucleosides such as purified 8-OH-dG,
8-OH-rGuo or the like obtained by the above purification method, it has high accuracy
and reproducibility.
[0045] The scope of the techniques of the present invention is not limited to the above
embodiments. Various modifications can be made without departing from the sprit or
scope of the present invention. For example, the composition of the eluent (solution
A and solution B) and the washing solution (solution C), or the like may be appropriately
modified corresponding to the columns (fillers) to be used.
[0046] The measuring method for oxidatively damaged guanine nucleosides of the present invention
may be used in individual carcinogenesis risk evaluation, prediction and diagnosis
of various disorders related to active oxygen (for example diabetes), evaluation of
degree of aging or general health.
[0047] The evaluation method for the results obtained by the measuring method, is described
below using an example in the case of 8-OH-dG. As well as the urine sample, 8-OH-dG
standard solution was injected into the analyzer periodically. Then, these peak areas
were compared to calculate the 8-OH-dG concentration in the sample. The calculated
8-OH-dG concentration was then divided by the concentration of a standard substance
such as creatinine, or calculated as the amount of 8-OH-dG in the urine for 24 hours.
Examples
[0048] Hereunder is a specific description of the present invention using examples, however
the present invention is not to be considered as limited to these.
(Example 1)
<Preparation of urine sample 1>
[0049] 1ml of human urine was placed in each of two Eppendorf tubes and frozen at -20°C.
The frozen urine was thawed and homogenized. 100µl of each homogenate was diluted
with the same volume of slightly acidic solution (composition; 96ml of 0.6mM sulfuric
acid and 4ml of acetonitrile). Consequently, 12µg of 8-OH-dG and 12µg of 8-OH-rGuo
were added. The pH was adjusted below 7 by adding 6.7µl of 2M sodium acetate (pH4.5).
The mixture was well stirred and centrifuged at 15,000 rpm for 5 minutes. The supernatant
was made the urine sample 1.
[0050] 10µl of the urine sample 1 prepared as described above was purified by an anion-exchange
column (particle diameter of filler (manufactured by Bio-Rad) 12µm, internal diameter
1mm, guard column length 4cm, and main column length 12cm). The separation pattern
is shown in FIG. 3. The separation pattern was obtained using a UV detector ("UV-8020"
manufactured by Tosoh Corp.) (absorption wavelength 254nm).
[0051] As shown in FIG. 3, 8-OH-dG was eluted at a fixed time after the 8-OH-rGuo elution.
Therefore, by monitoring the 8-OH-rGuo elution the accurate elution position of 8-OH-dG
could be ascertained so that the fraction containing 8-OH-dG could be reliably obtained.
(Example 2)
<Preparation of urine sample 2>
[0052] 1ml of human urine was placed in each of two Eppendorf tubes and frozen at -20°C.
The frozen urine was then thawed and homogenized. 100µl of each homogenate was diluted
with the same volume of slightly acidic solution (composition; 96ml of 0.6mM sulfuric
acid and 4ml of acetonitrile). Consequently, 12µg of 8-OH-rGuo was added. The pH was
adjusted below 7 by adding 6.7µl of 2M sodium acetate (pH4.5). The mixture was well
stirred and centrifuged at 15,000 rpm for 5 minutes. The supernatant was made the
urine sample 2.
<Purification by anion-exchange chromatography>
[0053] The human urine sample 2 was purified using an anion-exchange column to obtain a
fraction containing 8-OH-dG (34 to 41 min). 0.3mM sulfuric acid, 2% acetonitrile eluent
was used as a solution A. The column temperature was 65°C and the flow rate was 17µl/min.
The separation pattern is shown in FIG. 4. The anion-exchange column was made using
a filler of an Aminex HPX-72S column (manufactured by Bio-Rad) (300x7.8mm, particle
diameter 12µm, degree of cross-linkage 8%, and sulfate type) refilled in a column
of an internal diameter of 1mm (guard column length 4cm, and main column length 12cm).
The separation pattern was obtained using the UV detector ("UV-8020" manufactured
by Tosoh Corp.) (absorption wavelength 254nm).
<Purification by reverse phase chromatography>
[0054] 119µl of the fraction containing 8-OH-dG (34 to 41 min) obtained by the anion-exchange
chromatography was automatically injected into a reverse phase chromatography. A Shiseido
Capcell Pak C18 MG (S-5µm) (250x4.6mm) was used for the reverse phase column. 10mM
of phosphate buffer (pH6.7; the pH may slightly vary since it was prepared by diluting
0.1M phosphate buffer (pH6.7)) and 5% MeOH eluent (solution B) were used. The column
temperature was 40°C and the flow rate was 0.8ml/min. The separation pattern is shown
in FIG. 5. The separation pattern was obtained using an electrochemical detector ("ESA
Coulochem II" from ESA, Inc.) (voltage: 350mV in guard cell; 150mV at channel 1; 300mV
at channel 2).
(Example 3)
<Purification by anion-exchange chromatography>
[0055] Another example of a separation pattern of the human urine sample 2 is shown in FIG.
6. 20µl of human urine sample 2 was injected. As the separation condition, a column
MCI GEL CA08F (7µm) (internal diameter 1.5mm, guard column length 5cm, and main column
length 15cm) was used. The column temperature was 65°C, and the flow rate was 36µl/min.
0.3mM sulfuric acid, 2% acetonitrile eluent was used as a solution A. The separation
pattern was obtained using the UV detector ("UV-8020" manufactured by Tosoh Corp.)
(absorption wavelength 254nm).
(Example 4)
<Purification by reverse phase chromatography>
[0056] FIG. 7 shows another example where the fraction containing 8-OH-dG obtained by the
anion-exchange chromatography was analyzed by reverse phase chromatography. A YMC-Pack
ODS-AM (S-5µm) (250x4.6mm) was used for the reverse phase column. 10mM of phosphate
buffer (pH7.2; the pH may slightly vary since it was prepared by diluting 0.1M phosphate
buffer (pH7.2)) containing 5% MeOH (solution B) was used. The column temperature was
40°C and the flow rate was 0.9ml/min. The separation pattern was obtained using the
electrochemical detector ("ESA Coulochem II" from ESA, Inc.) (voltage: 350mV in guard
cell; 150mV at channel 1; 300mV at channel 2).
[0057] As is clear from FIG. 5 and FIG. 7, according to the method described above, no impurity
was found in the vicinity of the purified 8-OH-dG, showing superiority in the accuracy
of measuring 8-OH-dG.
(Example 5)
<Preparation of urine sample 3>
[0058] 1ml of rat urine was placed in each of two Eppendorf tubes and frozen at -20°C. The
frozen urine was thawed and homogenized. 100µl of each homogenate was diluted with
the same volume of slightly acidic solution (composition; 96ml of 0.6mM sulfuric acid
and 4ml of acetonitrile). Consequently, 12µg of 8-OH-rGuo was added. The pH was adjusted
below 7 by adding 6.7µl of 2M sodium acetate (pH4.5). The mixture was well stirred
and centrifuged at 15,000 rpm for 5 minutes. The supernatant was made the urine sample
3.
<Purification by anion-exchange chromatography>
[0059] The rat urine sample 3 was purified using an anion-exchange column to obtain a fraction
containing 8-OH-dG. 0.3mM sulfuric acid, 2% acetonitrile eluent was used as a solution
A. The column temperature was 65°C and the flow rate was 17µl/min. The anion-exchange
column was made using a filler of an Aminex HPX-72S column (manufactured by Bio-Rad)
(300x7.8mm, particle diameter 12µm, degree of cross-linkage 8%, and sulfate type)
refilled in a column of an internal diameter of 1mm (guard column length 5cm, and
main column length 15cm).
<Purification by reverse phase chromatography>
[0060] The fraction containing 8-OH-dG obtained by the anion-exchange chromatography was
automatically injected into and analyzed by a reverse phase column. The separation
pattern is shown in FIG. 8. A YMC-Pack ODS-AM (S-5µm) (300x4.6mm) was used for the
reverse phase column. 10mM of phosphate buffer (pH7.2; the pH may slightly vary since
it was prepared by diluting 0.1M phosphate buffer (pH7.2)) containing 5% MeOH (solution
B) was used. The column temperature was 40°C and the flow rate was 0.9ml/min. The
separation pattern was obtained using the electrochemical detector ("ESA Coulochem
II" from ESA, Inc.) (voltage: 350mV in guard cell; 150mV at channel 1; 300mV at channel
2).
(Example 6)
[0061] FIG. 9 shows another example where the fraction containing 8-OH-dG obtained from
rat urine by the anion-exchange chromatography (MCI, 7µm particle) was analyzed by
reverse phase chromatography. A Shiseido Capcell Pak C18 MG (S-5µm)(250x4.6mm) was
used for the reverse phase column. 10mM of phosphate buffer (pH6.0; the pH may slightly
vary since it was prepared by diluting 0.1M phosphate buffer (pH6.0)) containing 2%
MeOH (solution B) was used. The column temperature was 46°C and the flow rate was
0.75ml/min. The separation pattern was obtained using the electrochemical detector
("ESA Coulochem II" from ESA, Inc.) (voltage: 400mV in guard cell; 280mV at channel
1; 350mV at channel 2).
[0062] As is clear from FIG. 8 and FIG. 9, it was also possible to measure 8-OH-dG in the
rat urine sample 3.
INDUSTRIAL APPLICABILITY
[0063] According to the purification method by anion-exchange chromatography of the present
invention, oxidatively damaged guanine nucleosides such as 8-OH-dG, 8-OH-rGuo or the
like can be easily purified and recovered with a high recovery rate.
[0064] Moreover particularly in the purification of 8-OH-dG, by previously adding the 8-OH-rGuo
to the sample as the internal standard marker for 8-OH-dG, the accurate elution time
of 8-OH-dG can be obtained so that the fraction containing 8-OH-dG can be reliably
collected. Furthermore, since the flow rate of the anion-exchange column (HPLC-1)
in the first purification step is very low, the consumption of the eluent (solution
A) and the washing solution (solution C) is extremely small, and the amount of the
effluent waste after purification is small, so that the method is also preferable
from the aspect of environmental protection. Moreover, since the measuring method
of the present invention measures the purified oxidatively damaged guanine nucleosides
such as purified 8-OH-dG, 8-OH-rGuo or the like, obtained by the above purification
method, it has high accuracy and reproducibility. According to the 8-OH-dG analyzer
of the present invention, the anion-exchange column (HPLC-1) 11 specifically absorbs
8-OH-dG contained in the sample, increasing the recovery rate and removing almost
all impurities contained in the sample at once. Moreover, by continuous operation
a large amount of samples can be treated. Since the analyzer is relatively low in
price, it is superior in terms of economic efficiency.
1. A purification method for oxidatively damaged guanine nucleosides generated as a result
of guanine damage in DNA or RNA, comprising a first purification step for purifying
oxidatively damaged guanine nucleosides contained in a sample by anion-exchange chromatography.
2. A purification method for oxidatively damaged guanine nucleosides according to claim
1, wherein said oxidatively damaged guanine nucleoside is 8-hydroxydeoxyguanosine
(8-OH-dG).
3. A purification method for 8-hydroxydeoxyguanosines (8-OH-dG) contained in a sample,
wherein 8-hydroxyguanosines (ribonucleosides) (8-OH-rGuo) are previously added to
the sample as an internal standard marker for 8-OH-dG so as to purify it.
4. A purification method for 8-OH-dG (8-OH-dG) contained in a sample, wherein 8-hydroxyguanosine
(ribonucleosides) (8-OH-rGuo) is previously added to the sample, comprising a first
purification step for purifying said sample by anion-exchange chromatography, and
a second purification step for further purifying the fraction containing 8-OH-dG obtained
in the first purification step by reverse phase chromatography.
5. A purification method for oxidatively damaged guanine nucleosides according to claim
1 or claim 2, wherein said sample is urine.
6. A purification method for 8-hydroxydeoxyguanosines (8-OH-dG) according to claim 3
or claim 4, wherein said sample is urine.
7. A measuring method for oxidatively damaged guanine nucleosides comprising a measuring
step for measuring purified oxidatively damaged guanine nucleosides obtained by the
purification method of any one of claim 1, claim 2 and claim 5.
8. A measuring method for 8-OH-dG comprising a measuring step for measuring purified
8-hydroxydeoxyguanosines (8-OH-dG) obtained by the purification method of any one
of claim 3, claim 4 and claim 6.
9. A measuring method for 8-OH-dG according to claim 8, wherein said purified 8-hydroxydeoxyguanosines
(8-OH-dG) are measured in anion-exchange chromatography in the order of;
(1) peak recognition of ribonucleosides 8-OH-rGuo,
(2) starting of 8-OH-dG fractionation after a fixed time,
(3) finishing of 8-OH-dG fractionation after a fixed time, and
(4) optionally mixing 8-OH-dG fractions, and then injected into a reverse phase column.
10. An apparatus for purifying and measuring 8-hydroxydeoxyguanosines (8-OH-dG), comprising;
an anion-exchange column (HPLC-1) which specifically absorbs 8-OH-dG contained in
a sample,
a UV detector which detects an elution position of 8-hydroxyguanosine (ribonucleoside)
(8-OH-rGuo),
a reverse phase column (HPLC-2) which further purifies the fraction containing 8-OH-dG
obtained from the anion-exchange column (HPLC-1), and
a detector which measures the purified 8-OH-dG obtained from the reverse phase column
(HPLC-2).
11. A program for controlling a process for recovering 8-hydroxydeoxyguanosines (8-OH-dG)
contained in a sample by column chromatography, which executes on a computer processes
for:
receiving a peak signal of a marker (8-OH-rGuo) previously added to the sample from
a UV detector;
outputting a signal to open a valve connected to a sampler, during 8-OH-dG elution
after a fixed time;
starting fractionation; and
outputting a fractionation termination signal after another fixed time;
and then outputting a signal to inject the obtained 8-OH-dG fraction into a second
purifying column;
thereby purifying and recovering a detected substance (8-OH-dG) eluted from the column.